The goal of this project is to characterize by genetic, physical, and biochemical means the human adenovirus specified DNA binding protein (DBP). We will define genetically the interrelationships of the broadly defined functions associated with DBP (e.g. viral DNA replication, late gene turn on, and early gene shut off). Cell lines which complement one or all the different functions of DBP will be constructed by the cotransformation technique of Wigler et al. (1979). These lines will be used to isolate, propagate, and aid in the biological characterization of DBP mutants. DBP variants which have differential affects on the protein's various functions will be generated by general mutagenesis of adenovirus in vivo or site directed mutagenesis in vitro. The latter employ a new scheme which should allow a number of proven methods of site directed mutagenesis to be used on large genomes. Deletion mapping of existing as well as new DBP mutants will be used to construct a fine structure genetic map. This map will be essential in determining whether or not the DBP gene has separable functional domains. Having isolated and biologically characterized sets of mutants which differentially affect the various DBP functions, we hope to correlate the altered functions with changes in the physical and biochemical properties of the protein.